This project focuses on identifying and characterizing novel molecules and new mechanisms underlying craniofacial development and their relevance to tissue engineering, with particular focus on salivary and neural crest development. [unreadable] [unreadable] We are addressing the following major questions:[unreadable] 1. How do embryonic salivary glands and other tissues generate their large epithelial surface area during the process of branching morphogenesis? Specifically, how is cleft formation to delineate buds mediated and regulated? How can we facilitate bioengineering for organ replacement -- particularly of salivary glands -- by understanding branching morphogenesis and by developing reconstitution approaches? [unreadable] 2. What are the roles of cell motility versus extracellular matrix expression or remodeling and their regulation in branching morphogenesis and in other major tissue rearrangements such as cranial neural crest development? [unreadable] [unreadable] We are applying a variety of approaches to begin to answer these complex questions, including laser microdissection, gene expression profiling, RNA interference, whole-embryo, organ and cell culture, confocal immunofluorescence and video time-lapse microscopy, as well as a variety of functional inhibition and reconstitution approaches.[unreadable] [unreadable] Potential future clinical replacement of salivary gland function destroyed by radiation therapy for oral cancer or by Sjogrens Syndrome will be challenging, because it will require restoration of enough secretory epithelium to produce adequate volumes of salivary fluid to alleviate xerostomia (salivary hypofunction). This general biological problem of how to obtain sufficient surface area in compact organs for secretion is normally solved during embryonic development by the process of branching morphogenesis. During development, a single embryonic bud first develops clefts and buds. It then undergoes repetitive branching to provide the large surface areas needed for effective secretory output. Regardless of whether eventual clinical replacement will involve salivary regeneration or an artificial salivary gland, a major challenge is how to create numerous branched epithelial structures. We have been applying a variety of approaches to identify novel mechanisms, with a particular focus on extracellular matrix-cell interactions and dynamic movements of both cell and extracellular matrix that drive branching.[unreadable] [unreadable] We had previously established essential roles for fibronectin and its integrin receptor in salivary branching morphogenesis accompanied by substantial cell migration. Our unpublished studies also implicated actomyosin contractility in branching. We will continue to examine the underlying mechanisms and roles of this cell migration in branching morphogenesis. Our current hypothesis is that there are direct causal links between fibronectin expression with local matrix accumulation and the process of cleft extension to delineate buds, e.g. fibronectin-induced local expression of specific regulatory molecules. In order to search for such a novel regulator of morphogenesis, we applied our previously developed method termed T7-SAGE with laser microdissection to compare gene expression patterns of epithelial cells adjacent to clefts with those in buds. The goal was to identify genes that are differentially activated at each site. Besides fibronectin and TIMP3, each of which are now known to be needed for branching morphogenesis, we have found a previously uncharacterized gene present in cleft epithelial cells but not bud cells. We are examining whether it plays a role in branching morphogenesis by expression analyses using in situ hybridization and RT-PCR, as well as RNA interference approaches.[unreadable] [unreadable] A process of cell separation that is even more dramatic than cleft formation occurs during the scattering of cells at the initiation of cranial neural crest cell migration; these cells ultimately generate many of the tissues and structures of the face and mouth. We used ultra-micro array analysis to identify two dozen genes expressed 10-fold or higher in newly forming chick cranial neural crest compared to neural tube. We are focusing on strongly differentially expressed extracellular matrix proteins and are attempting to characterize a role for one of them in neural crest formation.